Telomerase
Telomerase is a ribonucleoprotein that catalyzes the addition of telomeric repeats to the ends of telomeres. Telomeres are long stretches of repeated sequences that cap the ends of chromosomes and are believed to stabilize the chromosome. In humans, telomeres are typically 7-10 kb in length and comprise multiple repeats of the sequence-TTAGGG-. Telomerase is not expressed in most adult cells, and telomere length decreases with successive rounds of replication. After a certain number of rounds of replication, the progressive shortening of the telomeres results in the cells entering a telomeric crisis stage, which in turn leads to cellular senescence. Certain diseases are associated with rapid telomeric loss, resulting in premature cell senescence. Expression of the gene encoding the human telomerase protein in human cells has been shown to confer an immortal phenotype, presumably though bypassing the cells' natural senescence pathway. In addition, expression of the telomerase gene in aging cells with short telomeres has been shown to produce an increase in telomere length and restore a phenotype typically associated with younger cells.
Somatic cells, in contrast to tumor cells and certain stem cells, have little or no telomerase activity and stop dividing when the telomeric ends of at least some chromosomes have been shortened to a critical length, leading to programmed cellular senescence (cell death). Since the loss of telomeric repeats in somatic cells, leading to senescence, is augmented by low telomerase activity, induction of telomerase activity, which has the effect of adding arrays of telomeric repeats to telomeres, thereby imparts to mortal somatic cells increased replicative capacity, and impart to senescent cells the ability to proliferate and appropriately exit the cell cycle upon repair of damaged tissue.
Potential therapeutic benefits of increased telomerase activity in somatic cells include, for example, treatment of AIDS, which is characterized by the early senescence of the cytotoxic T lymphocytes (CD8+ cells) which are responsible for killing infected CD4+ cells (see e.g. Dagarag et al., 2003); neuroprotection in Alzheimer's patients (see. e.g. Mattson, 2000); wound healing, and maintenance of explant cells, such as adrenocortical cells (see e.g. Thomas et al., 2000) or bone marrow or stromal/mesenchymal graft cells (see e.g. Simonsen et al., 2002). Full citations of these references appear below.
References discussing these and other characteristics of telomerase include:
Allsopp, R. C. et al., “Telomere shortening is associated with cell division in vitro and in vivo”, Exp. Cell Res. 220(1):194-200 (September 1995).
Allsopp, R. C. et al., “Telomerase is required to slow telomere shortening and extend replicative lifespan of HSC during serial transplantation”, Blood (e-publication) Mar. 27, 2003.
Bodnar, A. G. et al., “Extension of life-span by introduction of telomerase into normal human cells” Science 279(5349):349-52 (Jan. 16, 1998).
Cech, T. R. et al., U.S. Pat. No. 6,093,809 (Jul. 25, 2000).
Cech, T. R. et al., U.S. Pat. No. 6,166,178 (Dec. 26, 2000).
Cech, T. R. et al., U.S. Pat. No. 6,261,836 (July 2001).
Chiu, C. P. et al., “Replicative senescence and cell immortality: the role of telomeres and telomerase” Proc. Soc. Exp. Biol. Med. 214(2):99-106 (February 1997).
Dagarag, M. et al., “Differential impairment of lytic and cytokine functions in senescent human immunodeficiency virus type 1-specific cytotox T lymphocytes”, J. Virol. 77(5):3077-83 (March 2003).
Farwell, D. G. et al., “Genetic and epigenetic changes in human epithelial cells immortalized by telomerase”, American Journal of Pathology 156(5): 1537-47 (May 2000).
Fujimoto, R. et al., “Expression of telomerase components in oral keratinocytes and squamous cell carcinomas”, Oral Oncology 37(2):132-40 (February 2001).
Funk, Walter D. et al., “Telomerase expression restores dermal integrity to in vitro-aged fibroblasts in a reconstituted skin model”, Experimental Cell Research 258(2):270-278 (Aug. 1, 2000).
Hannon, G. J. and Beach, D. H., “Increasing proliferative capacity and preventing replicative senescence by increasing telomerase activity and inhibiting pathways inhibiting cell proliferation”, PCT Int. Appl. Pubn. No. WO 2000/031238 (June 2000).
Hannon, G. J. et al., Extension of cellular lifespan using telomerase-activating therapeutic agents”, PCT Int. Appl. Pubn. No. WO 99/35243 (July 1999).
Harle-Bachor, C. et al., “Telomerase activity in the regenerative basal layer of the epidermis inhuman skin and in immortal and carcinoma-derived skin keratinocytes”, Proc Natl Acad Sci USA 93(13):6476-81 (Jun. 25, 1996).
Harley, C. B. et al., “Telomeres shorten during ageing of human fibroblasts”, Nature 345(6274):458-60 (May 31, 1990).
Harley, C. B. et al., “Telomerase, cell immortality, and cancer”, Cold Spring Harb. Symp. Quant. Biol. 59:307-15 (1994).
Harley, C. B. et al., “Telomeres and telomerase in aging and cancer”, Curr. Opin Genet. Dev. 5(2):249-55 (April 1995).
Harley, C. B. et al., “Telomerase and cancer”, Important Adv. Oncol. 57-67 (1996).
Harley, C. B., “Human aging and telomeres”, Ciba Found. Symp. 211:129-39 (1997).
Harley, C. B., “Telomerase is not an oncogene”, Oncogene 21: 494-502 (2002)
Henderson, S. et al., “In situ analysis of changes in telomere size during replicative aging and cell transformation”, J. Cell Biol. 134(1):1-12 (July 1996).
Jiang, X. R. et al., PCT Pubn. No. WO 02/91999.
Jiang, X. R. et al., “Telomerase expression in human somatic cells does not induce changes associated with a transformed phenotype”, Nature Genetics 21(1): 111-4 (January 1999).
Kang, M. K. et al., “Replicative senescence of normal human oral keratinocytes is associated with the loss of telomerase activity without shortening of telomeres”, Cell Growth & Differentiation 9(1):85-95 (January 1998).
Kim, N. W. et al., “Telomerase activity assays”, U.S. Pat. No. 5,629,154 (May 1997).
Lee, K. M. et al., “Immortalization with telomerase of the Nestin-positive cells of the human pancreas”, Biochem Biophys Res Commun 301(4):1038-44 (Feb. 21, 2003).
Ludwig, A. et al., “Ribozyme cleavage of telomerase mRNA sensitizes breast epithelial cells to inhibitors of topoisomerase”, Cancer Res., 61: 3053-3061 (2001).
Mattson, M. P., “Emerging neuroprotective strategies for Alzheimer's disease: dietary restriction, telomerase activation, and stem cell therapy”, Exp Gerontol. 35(4):489-502 (July 2000).
Morales, C. P. et al., “Absence of cancer-associated changes in human fibroblasts immortalized with telomerase”, Nature Genetics 21(1): 115-8 (January 1999).
Oh, H. and Schneider, M. D., “The emerging role of telomerase in cardiac muscle cell growth and survival”, J Mol Cell Cardiol 34(7):717-24 (July 2002).
Simonsen. J. L. et al. “Telomerase expression extends the proliferative life-span and maintains the osteogenic potential of human bone mar-row stromal cells”, Nat Biotechnol 20(6):592-6 (June 2002).
Thomas, M., Yang, L., and Hornsby, P. J., “Formation of functional tissue from transplanted adrenocortical cells expressing telomerase reverse transcriptase”, Nat Biotechnol 18(1):39-42 (January 2000).
Vasa, M. et al., “Nitric oxide activates telomerase and delays endothelial cell senescence”, Circ. Res. 87(7):540-542 (2000).
Villeponteau, B. et al., U.S. Pat. No. 5,583,016 (December 1996).
West, M. D. et al., “Methods of screening for compounds that derepress or increase telomerase activity”, U.S. Pat. No. 6,007,989 (December 1999).
White, M. A., “Assembly of telomerase components and chaperonins and methods, and compositions for inhibiting or stimulating telomerase assembly”, PCT Int. Appl. Pubn. No. WO 2000/08135 (February 2000).
Yang, J. et al., “Telomerized human microvasculature is functional in vivo”, Nature Biotechnology (United States) 19(3):219-24 (March 2001).
Yang, J., et al., “Human endothelial cell life extension by telomerase expression”, J. Biol. Chem. 274(37):26141-8 (Sep. 10, 1999).
Yudoh, K. et al., “Reconstituting telomerase activity using the telomerase catalytic subunit prevents the telomere shorting and replicative senescence in human osteoblasts”, J. Bone and Mineral Res. 16(8):1453-1464 (2001).
Methods of increasing telomerase activity therapeutically have been investigated by, for example, Bodnar (1997), White (2000), Hannon et al. (1999; 2000), Franzese et al. (2001), and Yudoh et al. (2001), all cited above. In these reports, telomerase activity is generally increased by overexpression of hTRT, the gene encoding the protein component of human telomerase, or by expression of proteins which mediate assembly of telomerase, e.g. heat shock proteins (White). Franzese et al. reported that Saquinavir, a protease inhibitor prescribed for treatment of HIV infection, increased telomerase activity in peripheral blood mononuclear cells; Vasa et al. described activation of telomerase, and a resulting delay in endothelial senescence, by administration of a nitric oxide (NO) precursor.
Astragalosides and Ginsenosides
Compounds of the astragaloside and ginsenoside families have been reported as having various biological effects. References discussing biological activity of astragalosides and ginsenosides include:
Bedir, E. et al., “Immunostimulatory effects of cycloartane-type triterpene glycosides from Astragalus species”, Biol & Pharm Bull 23(7):834-7 (2000).
Binder, B. et al., “Use of triterpensaponins, such as notoginsenoside R1 (NR1) and/or astragaloside (ASIV) for preparing medicaments”, U.S. Pat. No. 5,770,578 (June 1998).
Calzada, L. et al., “Effect of tetracyclic triterpenes (argentatins A, B and D) on the estradiol receptor of hormone-dependent tumors of human breast”, Medical Science Research 23(12):815-16 (1995).
Chen, X. et al., “Protective effect of ginsenoside Rg1 on dopamin-induced apoptotis in PC12 cells”, Acta Pharmacol Sinica 22(8):673-678 (2001).
Hashimoto, K. et al., “Skin tissue regeneration promoters comprising ginsenoside Rb1”, WO 200192289 (2001); EP 1295893 A1 (2003).
Hong, H.-Y. et al., “Stimulatory effects of ginsenoside-Rg1 on p56lck kinase and cell proliferation in Jurkat T cells”, Korean J. Ginseng Sci. 19(2): 117-21 (1995).
Huang, Y. et al., “Selected non-timber forest products with medicinal applications from Jilin Province in China”, Conference Title: Forest communities in the third millennium: Linking research, business, and policy toward a sustainable non-timber forest product sector; Kenora, Ontario, Canada, 1-4 Oct., 1999; General Technical Report-North Central Research Station, USDA Forest Service (No. NC-217): p. 93-101 (2000).
Kaneko, M. et al., “Accelerated recovery from cyclophosphamide-induced leukopenia in mice administered a Japanese ethical herbal drug, Hochu-ekki-to”, Immunopharmacology 44(3):223-231 (1999).
Kinjo, J. et al., “Anti-herpes virus activity of fabaceous triterpenoidal saponins”, Biological & Pharmaceutical Bulletin 23(7):887-9 (July 2000).
Khushbaktova, Z. A. et al., “Influence of cycloartanes from plants of the genus Astragalus and their synthetic analogs on the contractive function of the myocarbium and the activity of Na, K-ATPase”, Chem. Nat. Compounds 30(4): 469-473 (1994).
Lee, Y. J. et al., “Ginsenoside-Rg1, one of the major active molecules from Panax ginseng, is a functional ligand of glucocorticoid receptor”, Mol Cell Endocrinol 133(2):135-40 (October 1997).
Liu, P. et al., “Effect of ginsenosides Rb1, Rg1, Rh1 and Re on proliferation of cells in vitro”, Tianraw Chanwzt Yanjiu Yu Kaifa 8(4):36-41 (1996); CA Abstract No. 1997:400846.
Oda, K. et al., “Adjuvant and haemolytic activities of 47 saponins derived from medicinal and food plants”, Biol. Chem. 381(1):67-74 (2000).
Pistelli, L., et al., “Antimicrobial and antifungal activity of crude extracts and isolated saponins from Astragalus verrucosus”, Fitoterapia 73(4):336-339 (2002).
Prince, R. L. and Min X., “Compositions and method for treating or preventing osteoporosis”, PCT Pubn. No. WO 2001/01996.
Sengupta, S. et al., “Pharmaceutically effective compounds and their use”, PCT Pubn. Nos. WO 2002/69980 and WO 2002/07732.
Wang, S. et al., “Promoting effect of ginsenoside Re on the proliferation of murine bone marrow cells”, Baiqiuen Yike Doxue Xuebao 23(2): 141-142 (1997); CA Abstract No. 1997:570234.
Wang, Y-P. et al., “Effect of astragaloside IV on T, B lymphocyte proliferation and peritoneal macrophage function in mice”, Acta Pharmacologica Sinica 23(3):263-6 (March 2002).
Yasukawa, K. et al., “Sterol and triterpene derivatives from plants inhibit the effects of a tumor promoter, and sitosterol and betulinic acid inhibit tumor formation in mouse skin two-stage carcinogenesis”, Oncology 48(1):72-6 (1991).
Yamamoto, M. et al., “The stimulatory effects of ginseng saponins on proliferation and DNA synthesis of human vascular endothelial cells and skin fibroblasts in relation to cytokines or growth factors”, Nissei Byoin Igaku Zasshi 24(1): 12-13 (1996).
Zhang W. J. et al., “Regulation of the fibrinolytic potential of cultured human umbilical vein endothelial cells: astragaloside IV downregulates plasminogen activator inhibitor-1 and upregulates tissue-type plasminogen activator expression”, Journal of Vascular Research 34(4):273-80 (July-August 1997).
Zi-Pu, L. and Qian, C., “Effects of astragaloside IV on myocardial calcium transport and cardiac function in ischemic rats”, Acta Pharmacol Sin 23(10): 898-904 (October 2002).